All chemicals, unless otherwise stated, were purchased from Sigma.
All animal experimental procedures were performed according to either the UK Animals (Scientific Procedures) Act of 1986 or according to the Italian law (art. 7, Legislative Decree No. 116, 27 January 1992), which acknowledged the European Directive 86/609/EEC, and GlaxoSmithKline policy on the care and use of laboratory animals and related codes of practice. All efforts were made to minimise the number of animals used.
All human material used in this research was collected and used in compliance with the Declaration of Helsinki and in accordance with any relevant laws. In all cases informed consent has been obtained from the donor or donor's next of kin.
HMIT nucleotide sequence [NH_001033633] was used to generate HMIT null-mutant mice.
The plasmids used were pcDNA3.1/V5-His-TOPO (Invitrogen) containing rat wild-type or rat HMIT triple mutant DNAs, all generated at GlaxoSmithKline. HMIT was cloned in frame with the V5-His tags by deleting the stop codon. The triple mutant, as described previously [7
], was generated by site-directed mutagenesis using the QuikChange®
Multi Site-Directed Mutagenesis Kit (Stratagene) following manufacturer's instructions.
HMIT antibody generation
Rabbit polyclonal affinity purified antibody anti-HMIT #21 (anti-HMIT:21) was produced by Open Biosystems. The following conserved peptide (human, rat and mouse) coupled to KLH was chosen: RWLIQKGQTQKARRILS [rat accession number AJ315643], which is located in the intracellular loop between transmembrane domains 6 and 7.
Cell culture and transient transfection
Human embryonic kidney (HEK) 293 cells were cultured in MEM (Invitrogen), 10% (v/v) foetal bovine serum (FBS) (Invitrogen), 1% (v/v) MEM non-essential amino-acids (Invitrogen), 2% (v/v) L-glutamine (Invitrogen).
Cells were seeded at 30,000–50,000 cells per well (in 24-well plate format) or at 200,000 cells per well (in 6-well plate format) and grown for 24 h before transfection using Fugene 6 (Roche) according to manufacturer's instructions. Cells were fixed or lysed 48 h post-transfection.
Rat and mouse cortical neurone preparation
Cortical neurones were cultured from gestational day 18 rat foetal tissue of Sprague Dawley rats [9
] or gestation day 15 mouse foetal tissue. Briefly, cerebral cortices were collected in Hank's balanced salts solution, the meninges removed, and cortex dissected and dissociated with a papain tissue dissociation kit (Worthington) following the manufacturer's instructions. Cells were resuspended in Neurobasal medium (it contains 40 μM inositol) containing 2% (v/v) B27 supplement, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin and 50 μg/ml streptomycin (media components from Invitrogen).
Rat hippocampal slice preparation
Sprague-Dawley rats (postnatal day (P) 16–27) were deeply anaesthetised with isoflurane by inhalation and the brains were removed following decapitation. Using a vibratome (HM650V, Carl Zeiss Ltd.), horizontal hippocampal slices (250–300 μm thick) were cut in ice-cold artificial cerebrospinal fluid (aCSF) of the following composition: 125 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4.H2O, 25 mM glucose, 1 mM CaCl2, 2 mM MgCl2; bubbled with 95% O2/5% CO2. Slices were incubated at room temperature in aCSF for an hour before experimentation and used for up to 8 hours after preparation.
Intracellular myo-inositol measurement (Table )
800,000 cortical neurones/well were plated in 6-well plates. After 8–9 days in culture the neurones were stimulated with inositol, lysed in 500 μl of ice-cold acetonitrile with 100 mM ammonium acetate (90:10, v/v), briefly spun down and supernatant analysed. A standard curve was generated using pure myo-inositol (10–0.1 μM). A Jasco binary gradient HPLC system was used. Eluates were detected using an Applied Biosystems Sciex API-4000 triple-quadrupole mass spectrometer equipped with a TurboIonSpray ion-source. The operating parameters of the ion-source, including analyte-dependent and source-dependent were optimised to obtain the optimum performance from the mass spectrometer for the analysis of myo-inositol. The sensitivity of detection for myo-inositol in the negative ion mode was found to be higher than in positive mode. The collision associated dissociation of myo-inositol precursor at m/z (mass/charge) 179 produced abundant ions at m/z 161, 117, 99 and 87. Of these product ions m/z 179→87 transition was selected as it provided the greatest selectivity. For [2H6]-myo-inositol (CDN isotopes) the best signal for precursor-to-product transition was m/z 185→167. The optimum values for declustering potential, collision energy, entrance potential and collision exit potential for the precursor-to-product ion transitions selected are listed below. The source dependent parameters for myo-inositol consisted of collision gas, curtain gas, ion spray gas 1 and 2, ionspray voltage and the temperature of the heater gas, with optimum values of 6, 10, 20, 30, -4.5 kV and 600°C, respectively. An aliquot (5 μl) of myo-inositol was loaded onto a 150 × 2.1 mm i.d. Luna HILIC column (Phenomenex). The column temperature was maintained at 35°C. A linear gradient elution profile was used for the separation of myo-inositol. The mobile-phase consisted of eluent 'A', composed of a mixture of acetonitrile and 100 mM ammonium acetate buffer (90:10%, (v/v)), and eluent 'B', composed of acetonitrile, water and 100 mM ammonium acetate buffer (50:40:10%, (v/v)). A flow rate of 0.43 ml/min was used. The following elution profile was used: 0.0 min – 100% A; 2.5 min – 100% A; 12.0 min – 50% A and 50% B (linear gradient from 2.5 to 12.0 min); 12.9 min – 50% A and 50% B; 13.0 min – return to 100% A; hold for 7 min before proceeding to the next injection.
Intracellular myo-inositol measurement
Immunocytochemistry – primary neurones and cell lines
Cells were seeded at 30,000–50,000 cells per well in 24-well plate format on glass coverslips coated with poly-D-lysine. Following transfection, cells were fixed with 4% (w/v) paraformaldehyde (PFA) for 15 min at room temperature (RT). Cells were washed three times with PBS and permeabilised with 0.3% (v/v) Triton X100 for 5 min. 5% (v/v) normal goat serum (Chemicon) in PBS was used as blocking agent for 1 h. Primary antibodies were diluted in blocking solution and cells incubated overnight at 4°C with gentle shaking, followed by three washes with PBS. The secondary antibodies used were Alexa Fluor conjugated antibodies (Molecular Probes; 1:400), applied for 1 h at RT (in darkness). The coverslips were mounted onto glass slides with ProLong Gold antifade reagent with or without DAPI (Molecular Probes) to detect the cell nuclei. Cells were visualised using an Olympus BX51 microscope equipped with epifluorescent optics using Image-Pro Plus (Media Cybernetics) or Special Cell F (Olympus) software to analyse the images. For confocal imaging a Leica TLS SP confocal microscope with 63× and 100× water objectives was used with Leica confocal software.
Immunohistochemistry – rat and human brain
The analysis of the HMIT protein distribution in rat brain was performed on adult animals anaesthetised with chloral hydrate (400 mg/kg) and transcardially perfused with saline for 5 min, followed by 15 min of fixation with 4% (w/v) PFA in PBS. Thirty μm-thick coronal brain sections were cut using a vibratome (Leica) and left free-floating in PBS. The slices were firstly exposed to antigen-retrieval, consisting of 30 min incubation at 80°C in 0.1 M Tris-HCl added with 1 mM EDTA, pH 9 and then blocked for 1 h in PBS supplemented with 3% (v/v) goat serum (Vector Laboratories), 0.3% (v/v) Triton X-100 (Sigma) and 0.02% (v/v) bovine serum albumin (BSA) at RT. The slices were incubated overnight at 4°C in the same buffer with anti-HMIT:21 at a dilution of 1:200 and then incubated in PBS with the fluorescent secondary antibody 488 goat anti-rabbit (Alexa-Fluor; Molecular Probes). Finally, slices were mounted on slides with Vectashield-mounting medium for fluorescence (Vector Laboratories), covered with a coverslip and visualised by fluorescent microscopy.
The distribution of the HMIT protein in the human brain was investigated in formalin-fixed and paraffin embedded (FFPE) tissues obtained from Zoion, Inc. as an anatomical gift, following approved ethical guidelines. Five μm-thick tissue slices were cut using a microtome (Micron) and collected on slides. The tissue sections were then processed through a de-paraffination consisting of a series of washes in xylene, mixtures of xylene and ethanol, and decreasing ethanol concentrations (100%, 95%, 70%, 50%). The sections were then exposed to heat-induced antigen retrieval in a microwave oven (2 cycles of 5 min with an output of 700 W with a 1 min interval between the two boiling cycles) in 0.1 M Tris-HCl with 1 mM EDTA, pH 9. The slices were then incubated for 10 min in 1% (v/v) H2O2 in PBS containing 0.05% (v/v) Tween 20 and then blocked for 1 h at RT in the same blocking buffer used for rat tissues. In the same buffer, the slices were incubated overnight at 4°C with anti-HMIT:21 antibody at a dilution of 1:200. Subsequently, tissue sections were incubated with biotinylated anti-rabbit IgG (H + L) affinity purified made in goat secondary antibody (Vector Laboratories) in PBS with 1% (v/v) goat serum and then exposed for 45 min to avidin-biotinylated peroxidase complex (ABC Standard, Vector Laboratories), before incubation in 3,3'-diaminobenzidine (DAB, Vector Laboratories) for 3 min followed by a series of dehydrating washes (increasing ethanol concentrations and xylene).
[3H]-myo-inositol uptake in HEK293T cells
The method utilised was based on Uldry et al.
]. HEK293T cells were seeded at 40,000 cells per well in 24-well poly-D-lysine-coated plate format. 24 h after seeding, cells were transiently transfected with HMIT triple mutant DNA using Fugene 6 (Roche) according to the manufacturer's instructions. 48 h after transfection, [3
-inositol uptake was measured at pH 6.0 in a total assay volume of 500 μl. Cells were washed twice with K5 buffer (5 mM KCl, 127 mM NMDG, 10 mM D-glucose, 1 mM MgCl2
, 20 mM HEPES, 2.7 mM CaCl2
; pH 7.4). Cells were pre-incubated for 10 min at 37°C with 0, 1 or 3 mM phloridzin, a non-selective HMIT inhibitor, in 450 μl of K5 buffer (5 mM KCl, 127 mM NMDG, 10 mM D-glucose, 1 mM MgCl2
, 20 mM HEPES, 2.7 mM CaCl2
; pH 6.0). Phloridzin was prepared as a 100× stock in neat ethanol, subsequently diluted in the assay buffer to give a final concentration of 1% ethanol. The reaction was started by the addition of 50 μl (5 μCi) of [3
-inositol (GE Healthcare)/2 mM cold myo
-inositol mix, prepared in pH 6.0 K5 buffer. After 2 min the reaction was stopped by aspiration of the [3
-inositol and washing five times with 0.75 ml of ice-cold PBS. Cells were solubilised in 0.5 ml of 5% (w/v) sodium dodecyl sulfate (SDS) at 37°C for 30 min. Lysates were mixed with 4.5 ml of Ultima Gold XR (Perkin Elmer) scintillation liquid and radioactivity counted by scintillation spectroscopy (Tri-Carb 2800TR, Perkin Elmer). All data are reported as mean ± S.D.
[3H]-CDP-DAG accumulation in mouse HMIT null-mutant cortical neurones
Mouse HMIT null-mutant and wild-type cortical neurones, prepared as described above, were seeded at 150,000 cells per well in 24-well poly-D-lysine-coated plates. Seven or nine days after plating, [3
H]-CDP-DAG accumulation was measured using a method adapted from Atack et al.
]. Media was removed and neurones pre-incubated with 300 μl/well of modified Krebs-Hensleit buffer (118 mM NaCl, 25 mM NaHCO3
, 4.7 mM KCl, 1.3 mM CaCl2
, 1.2 mM MgSO4
, 1.2 mM KH2
, 5 mM HEPES, 10 mM glucose; pH 7.4) containing 0.3 μCi [3
H]-5-cytidine (Sigma) for 1 h at 37°C. Endogenous muscarinic G-protein coupled receptors were stimulated with 1 mM CCh in the presence of the inositol monophosphatase and inositol polyphosphate 1-phosphatase inhibitor LiCl (10 mM), to prevent formation of myo
-inositol, thus, resulting in [3
H]-CDP-DAG accumulation. Experiments were performed over a time course in the absence or presence of 3 mM extracellular myo
-inositol. Incubations were terminated by the addition of 1 ml of a mixture of methanol and chloroform (2:1, (v/v)) containing 1 M HCl to each well, which was left to extract for 5 min at RT. Well contents were transferred to centrifuge tubes; 310 μl of chloroform and 560 μl of 0.1 M HCl were added to aid phase separation. Samples were centrifuged at 0.1 rcf for 10 min at RT. An aliquot (400 μl) of the lower organic phase was removed, without disturbing the aqueous upper layer, transferred to scintillation vials and solvent was evaporated overnight. Ultima Gold XR scintillant liquid (4.5 ml) was then added and the samples counted by scintillation spectroscopy. All data are reported as mean ± S.D.
Whole-cell patch-clamp recordings
HEK293 cells and cultured rat neurones, prepared as above, were perfused with an external solution containing: 145 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 10 mM glucose, 2 mM CaCl2, 1 mM MgCl2, pH 7.4 with NaOH. In some experiments, NaCl was replaced with NMDG-Cl. Patch pipettes (5–10 MΩ tip resistance) were filled with: 135 mM KMeSO4, 10 mM HEPES, 4 mM NaCl, 4 mM Mg-ATP, 0.2 mM Na2GTP, 0.5 mM EGTA; pH 7.3; 280–300 mOsmol/kg. Membrane currents were recorded by whole-cell voltage-clamp using an Axopatch 200B amplifier and pClamp9 software (Molecular Devices). The holding membrane potential was set at -60 or -70 mV unless specified otherwise. Solutions containing test compounds were applied via a dual-barrel fast perfusion system (RSC-160; Biologic) and all experiments were conducted at RT.
Hippocampal slices were superfused (2–3 ml/min) at RT with aCSF: 125 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4.H2O, 25 mM glucose, 2 mM CaCl2, 1 mM MgCl2; pH 7.3 when bubbled with 95% O2/5% CO2. Interneurones in the CA3 stratum radiatum and CA3 and CA1 stratum lacunosum-moleculare were visualised under IR-DIC optics (Nikon). Membrane currents were recorded by whole-cell voltage-clamp using a Multipatch 700B amplifier and pClamp9 software (Molecular Devices). Patch pipettes had tip resistances of 5–10 MΩ when filled with internal solution: 130 mM KMeSO4 or CsMeSO4, 4 mM NaCl, 10 mM HEPES, 0.5 mM EGTA, 4 mM Mg-ATP, 0.2 mM Na2GTP; pH 7.3, 300 mOsmol/kg. Pressure micro-application of myo-inositol (5–15 p.s.i.; Picospritzer, Warner Instruments) was achieved using a glass pipette of tip diameter 50–150 μm positioned adjacent to the target cell body. All test compounds were bath perfused via gravity feed. In some experiments, brain slices were stimulated by: perfusion of glutamate (10 or 100 μM) for 5–30 min; phorbol 12-myristate 13-acetate (PMA) (0.5 or 5 μM) for 30–60 min; D-myo-phosphatidylinositol 4,5-bisphosphate-sn-1,2-di-O-octanoyl-glyceryl, 3-O-phospho linked (DiC8) (10 μM in the intracellular solution allowed to dialyse for at least 15 min before recording); 25 mM K+ external solution for 2 min; pre-incubation of slices at 30°C (for >2 h before recording); or depolarisation by current injection in current-clamp mode.
Data analysis of agonist-evoked currents was performed using pClamp9 (Molecular Devices) and Origin7.5 (Original Lab Corp.) software. Concentration-response curves were fitted with the Hill equation: y = 1+ (EC50/x)nH, where y is the membrane current, EC50 is the concentration of half-maximal efficacy, x is the agonist concentration, and nH is the Hill coefficient. All data are reported as mean ± S.E.M.
HMIT null-mutant mouse generation
The technical strategy for the KO mouse generation is described in Additional files 1